Reduced platelet numbers and function are common causes of pathologic bleeding, whereas increased platelet numbers and reactivity are believed to contribute to pathologic thrombosis. Platelet gene and protein expression - and hence, platelet production and reactivity - are largely controlled by megakaryocyte (Meg) transcription, post-transcriptional processing and translation. The expression of most protein-coding genes is regulated by microRNAs (miRNAs) that target mRNAs for degradation or inhibition. MiRNAs are important in all human physiology, and although dysregulated miRNA biology contributes to hematologic diseases, little is known about miRNA effects in Megs. We have measured platelet phenotypes, miRNA profiles and mRNA profiles on 154 healthy subjects. The phenotypic variation in this data allowed us to define a small number of specific master miRNAs regulating platelet reactivity through GPVI, PAR1, PAR4 and P2Y12. We hypothesize that Meg miRNAs regulate mRNAs that control platelet reactivity and platelet number. The overall goal of this research is to understand the function of miRNAs in human Megs (hMegs). Aim 1 will characterize the functionality of candidate master miRNAs regulating platelet aggregation. Candidate miRNAs will be transduced into hMegs differentiated from CD34+ stem cells and tested for agonist-induced cell activation. Predicted mRNA binding sites will be validated and function of novel mRNA targets tested in Megs. MiRNAs are established regulators of apoptosis genes in other cells, but miRNAs have no known role in regulating hMeg apoptosis or apoptosis-regulated thrombopoiesis. In additional preliminary data we have identified specific miRNAs associated with platelet number that target and knock down mRNAs encoding apoptosis-regulating genes also associated with platelet number (including BCL2L2 [Bcl-w], MCL1, CASP1 and others). Aim 2 will assess the role of miRNAs in Meg apoptosis and thrombopoiesis. Candidate miRNAs will be tested in hMegs for proplatelet formation, platelet production, Meg apoptosis and Meg mitochondrial function. Additional preliminary data indicates that blood cell type-preferential expression of some miRNAs can dictate cell-preferential transgene expression. Aim 3 will assess whether hematopoietic progenitor-preferential expression of miRNAs contributes to Meg-preferential gene expression. MiRNA levels will be profiled from laser-capture micro-dissection of primary human bone marrow cells, as well as peripheral blood cell lineages from the same subject. Binding sites for cell-preferential miRNAs will be engineered into lentiviral vectors, which will be infect hematopoietic stem cells and transgenes expression assessed in Megs and other lineages. The experiments in this proposal will clarify the role of miRNAs in Meg/platelet function and thrombopoiesis, improve our understanding of molecular mechanisms of lineage differentiation and platelet life span, and lay groundwork for miRNAs as biomarkers and potential therapeutic tools for altering Meg gene expression in a lineage-restricted manner in disorders of thrombosis and hemostasis.